Tomomi Fujii

ROS generation via NOX4 and its utility in the cytological diagnosis of urothelial carcinoma of the urinary bladder

BackgroundReactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. In the present research, we examined the pathobiological role of NADPH oxidase (NOX)4-mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology.MethodsNOX4 gene was silenced in vivo and in vitro by NOX4 siRNA transfection with or without atlocollagen. Cell cycle and measurement of ROS were analyzed by flowcytometry. Orthotopic implantation animal model was used in vivo experiment. NOX4 expression in urothelial carcinoma cells was observed by immunohistochemical analysis using surgical specimens of human bladder cancer. Urine cytology was performed after treatment with ROS detection reagents in addition to Papanicolaou staining.ResultsNOX4 was overexpressed in several UC cell lines and the NOX inhibitor, diphenylene iodonium reduced intracellular ROS and induced p16-dependent cell cycle arrest at the G1 phase. Moreover, silencing of NOX4 by siRNA significantly reduced cancer cell growth in vivo as assessed in an orthotopic mouse model. Immunohistochemistry demonstrated high expression of NOX4 in low grade/non-invasive and high grade/invasive UC including precancerous lesions such as dysplasia but not in normal urothelium. Then, we assessed the usefulness of cytological analysis of ROS producing cells in urine (ROS-C). Urine samples obtained from UC cases and normal controls were treated with fluorescent reagents labeling the hydrogen peroxide/superoxide anion and cytological atypia of ROS positive cells were analyzed. As a result, the sensitivity for detection of low grade, non-invasive UC was greatly increased (35% in conventional cytology (C-C) vs. 75% in ROS-C), and the specificity was 95%. Through ROS-C, we observed robust improvement in the accuracy of follow-up urine cytology for cases with previously diagnosed UC, especially in those with low grade/non-invasive cancer recurrence (0% in C-C vs. 64% in ROS-C).ConclusionsThis is the first report demonstrating that ROS generation through NOX4 contributes to an early step of urothelial carcinogenesis and cancer cell survival. In addition, cytology using ROS labeling could be a useful diagnostic tool in human bladder cancer.

Syndecan‐1 (CD138) contributes to prostate cancer progression by stabilizing tumour‐initiating cells

Increasing evidence suggests that tumour‐initiating cells (TICs) contribute to the development of prostate cancer. Here, we identified syndecan‐1 as a key molecule maintaining the stability of prostate cancer TICs. Holoclones harbouring the biological properties of stemness were derived from single‐cell cultures of the PC3 human prostate cancer cell line. These holoclones over‐expressed syndecan‐1, but showed reduced expression of NADPH oxidase (NOX) and synthesis of hydrogen peroxide and oxygen radicals. Stable RNA‐mediated silencing of syndecan‐1 gene expression up‐regulated NOX‐dependent generation of reactive oxygen species and reduced the survival of holoclones in vitro. Syndecan‐1 down‐regulation also strongly reduced the number of CD133+/CD44+ primitive cancer cells and tumour growth in vivo. Interestingly, syndecan‐1 gene knockdown significantly enhanced the tumour‐suppressive effects of docetaxel by inhibiting the docetaxel‐induced increase in CD133+/CD44+ cells in vivo. In the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, early intervention with a syndecan‐1 inhibitor (OGT2115) or syndecan‐1 RNAi reduced the incidence of adenocarcinoma and the number of c‐kit+/CD44+ cells in cancer foci. Finally, we found that syndecan‐1 immunopositivity in prostate cancer cells was significantly associated with biochemical recurrence after radical prostatectomy. Taken together, our results show that syndecan‐1 contributes to prostatic carcinogenesis by maintaining TICs and may be a target molecule for therapy. Copyright

Immunohistochemical analysis of inflammatory cells in benign and precancerous lesions and carcinoma of the prostate.

Objective: Inflammation is an important cause of tumorigenesis in various types of malignancy. Mediators derived from inflammatory cells are associated with cancer proliferation, angiogenesis, and DNA damage. In the present study, we immunohistochemically examined the infiltration patterns of inflammatory cells in benign glands including glandular hyperplasia, and in prostatic intraepithelial neoplasia and adenocarcinoma. Methods: Formalin-fixed, paraffin-embedded tissues were obtained from 100 patients with prostate cancer. All patients underwent radical prostatectomy. We assessed the number of infiltrating T cells (CD3+), B cells (CD20+, CD79alpha+), and macrophages (CD68+, CD204+) in benign and malignant prostate tumors. Results: CD68+ macrophages infiltrated benign glands to a higher extent than those of adenocarcinoma. In contrast, the number of CD204+ cells was higher in malignant glands than in benign glands. There was no significant difference in the number of infiltrating T cells between benign and malignant tumors; however, the number of infiltrating B cells was significantly reduced in malignant glands. Conclusions: Inflammation of the prostate may act on prostate carcinomas; particularly that involving M2 macrophage infiltration may play a significant role in prostate carcinogenesis.

ALKBH2, a novel AlkB homologue, contributes to human bladder cancer progression by regulating MUC1 expression

The ALKBH family of proteins are highly expressed in various types of human cancer where they are involved in tumor growth and progression. However, multiple isoforms of ALKBH exist and the effect of individual isoforms on the development of urinary bladder cancer is unknown, particularly the molecular mechanisms involved in the progression from a noninvasive to invasive phenotype. We examined the role and function of ALKBH2 in human bladder cancer development in vitro and provide the first report that suppression of ALKBH2 in a human urothelial carcinoma cell line, KU7, reduces the expression of the transmembrane mucin protein, MUC1, and induces G1 cell cycle arrest. Moreover, reduction of ALKBH2 suppressed epithelial to mesenchymal transition (EMT) via increasing E‐cadherin and decreasing vimentin expression. Transfection of MUC1 siRNA inhibited cell proliferation and EMT to the same extent as ALKBH2 gene silencing in vitro. ALKBH2 knockdown significantly suppressed MUC1 expression and tumor volume of bladder cancers in vivo as assessed in an orthotopic mouse model using ALKBH2 shRNA transfected KU7 cells. Immunohistochemical examination showed high expression levels of ALKBH2 in human urothelial carcinoma samples, especially in high‐grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)), and in carcinoma in situ but not in normal urothelium. This study demonstrates that ALKBH2 is an upstream molecule of the oncoprotein, MUC1, and regulates cell cycle and EMT, resulting in progression of urothelial carcinomas.

ALKBH3 Contributes to Survival and Angiogenesis of Human Urothelial Carcinoma Cells through NADPH Oxidase and Tweak/Fn14/VEGF Signals

Purpose: The role and function of a novel human AlkB homologue, ALKBH3, in human urothelial carcinoma development were examined. Experimental design: Biologic roles of ALKBH3 were examined by gene silencing analysis using in vitro and in vivo siRNA transfection. Immunohistochemical analyses of ALKBH3 and the related molecules using human bladder cancer samples were conducted to estimate the association with clinicopathologic or prognostic parameters. Results: ALKBH3 knockdown induced cell cycle arrest at the G1 phase through downregulation of NAD(P)H oxidase-2 (NOX-2)–mediated generation of reactive oxygen species (ROS). ALKBH3 knockdown reduced VEGF expression by reducing expression of tumor necrosis factor-like weak inducer of apoptosis (Tweak) and its receptor, fibroblast growth factor-inducible 14 (Fn14). Silencing of ALKBH3 or Tweak significantly suppressed invasion and angiogenesis of urothelial carcinoma in vivo as assessed both by a chorioallantoic membrane assay and in an orthotopic mouse model. Interestingly, not only urothelial carcinoma cells but also vascular endothelial cells within cancer foci expressed Fn14, which was strongly reduced by ALKBH3 and Tweak knockdown in vivo, suggesting that ALKBH3-dependent expression of Tweak stabilizes Fn14. Immunohistochemical examination showed high expression of ALKBH3, Tweak, and Fn14 in urothelial carcinoma, especially in high-grade, superficially, and deeply invasive carcinomas; moreover, Fn14-positive vessel counts within cancer foci were increased in invasive phenotypes. Conclusions: ALKBH3 contributes to development of urothelial carcinomas by accelerating their survival, angiogenesis, and invasion through NOX-2-ROS and Tweak/Fn14-VEGF signals. Clin Cancer Res; 18(19); 5247–55. ©2012 AACR.

Syndecan-1 up-regulates microRNA-331-3p and mediates epithelial-to-mesenchymal transition in prostate cancer

MicroRNAs (miRNAs) are small noncoding RNAs with a length of approximately 19–24 nucleotides that regulate gene expression through translational inhibition and contribute to the progression of various tumors including prostate cancer. Aberrant expression of miRNAs has been implicated in the progression and metastasis of prostate cancer. The present study aimed to investigate whether miR‐331‐3p controlled by syndecan‐1 positively affects the epithelial‐to‐mesenchymal transition (EMT). Overexpression of miR‐331‐3p upregulated mesenchymal markers such as vimentin, N‐cadherin, and snail and downregulated epithelial markers such as E‐cadherin and desmoplakin in the prostate cancer cell line PC3. We identified Neuropilin 2 and nucleus accumbens‐associated protein 1 as putative target molecules in silico, as they were closely associated with the expression of miR‐331‐3p and TGF‐β/Smad 4 signals. In situ hybridization and immunohistochemistry of radical prostatectomy samples revealed miR‐331‐3p in cancer cells with high Gleason patterns, in which EMT was demonstrated by decreased E‐cadherin, and increased vimentin staining. Syndecan‐1 gene silencing decreased levels of Dicer, which is involved in miRNA maturation. MiR‐331‐3p‐mediated miRNA maturation and enhanced EMT via effects on TGF‐β/Smad 4 and Dicer are essential for the development of prostate cancer mediated by syndecan‐1.

MicroRNA-331-3p Suppresses Cervical Cancer Cell Proliferation and E6/E7 Expression by Targeting NRP2

Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3′-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects.

Evaluation of RNA and DNA extraction from liquid-based cytology specimens.

Molecular diagnosis using DNA and RNA derived from malignant tumors and molecular biological tools such as the quantitative polymerase‐chain‐reaction (qPCR) is a commonly used technique in clinical pathology. In this report, we compared the qualitative extraction of RNA and DNA from cancer cells fixed using several liquid‐based cytology (LBC) kits. Ten to 1,000 cells from the T24 urinary bladder cancer cell line and SKG‐II cervical cancer cell line were fixed with 55% methanol and three different methanol‐based LBC solutions. The mRNA levels of CD44 in T24 cells and E7 in SKG‐II cells and DNA levels of p53 in T24 cells and E7 in SKG‐II cells were analyzed by qPCR. mRNA and DNA extracted from T24 and/or SKG‐II cells fixed with methanol‐based LBC solutions were efficiently detected, but to differing degrees, by qPCR. mRNA, and DNA from cells fixed with a formaldehyde‐containing fixative liquid were detected at significantly low copy numbers by qPCR. Our results demonstrate that LBC systems are powerful tools for cytopathology and immunocytochemistry applications. However, the appropriate fixative must be selected for cell preservation when a small number of LBC samples is used for molecular testing, particularly in RNA‐based molecular analyses. Diagn. Cytopathol. 2016;44:833–840.

Ubiquilin2 as a novel marker for detection of urothelial carcinoma cells in urine

Ubiquilin 2 (UBQLN2), an ubiquitin‐related protein, is strongly expressed in urothelial carcinoma cells, in contrast to no or less expression in non‐neoplastic cells; it protects cancer cells from reactive oxygen species (ROS)‐induced cytotoxicity. In this study, we investigated whether UBQLN2 immunostaining, using liquid‐based cytology sample could improve the accuracy of cytological urine diagnosis.

Phyllodes tumor of the prostate

In this report, we describe a case of phyllodes tumor of the prostate with a high value of prostate‐specific antigen (PSA). A 47‐year‐old man with symptoms of hematospermia presented with a steadily elevated serum PSA value of 60.76 ng/mL (normal range, <4 ng/mL). A needle biopsy revealed atypical stromal cells without any evidence of malignancy. After radical prostatectomy, the tumor measured 2.9 cm in diameter and consisted of a single nodule composed of irregular, elongated epithelial ducts and atypical stromal cells with enlarged, occasionally multinucleated, pleomorphic, or hyperchromatic nuclei. Immunohistochemistry showed that the atypical stromal cells were positive for vimentin, androgen receptor, estrogen receptor, progesterone, and 5α‐reductase, but negative for MIB‐1, PSA, SMA, p53, desmin, CD34, c‐kit, CD10, S‐100, and EGFR. Excess PSA might be secreted by hyperplastic luminal cells driven by 5α‐reductase‐positive stromal and epithelial cells. Array‐comparative genomic hybridization (array CGH) for genomic alterations revealed a gain of 11p13, which includes the WT1 gene, and a loss of 1p36.23 and 12p12.1. After surgery, the serum PSA value rapidly decreased to within the normal range; no recurrence or distant metastasis was noted after 2 years of follow up.